Purification and characterization of butyrylcholine-hydrolyzing enzyme from Pseudomonas polycolor.

A butyrylcholine-hydrolyzing enzyme (EC 3.1.1.-) fo Pseudomonas polycolor IFO 3918 was purified approximately 9270-fold with a recovery of 9.9% by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoretic and ultracentrifugational analyses. The molecular weight was determined as approximately 59000 by gel filtration. Isoelectric focusing electrophoresis revealed that the enzyme had an isoelectric point around pH 5.1. The enzyme catalyzed the hydrolysis of butyrylcholine with the miximum activity among various esters tested, and split benzoylcholine, propionylcholine and some aliphatic esters, but did not attact acetylcholine. The estimated value of Km at pH 7.5 and 25 degrees C was 7-10(-4) M for butyrylcholine. The enzyme was irreversibly inhibited by organophosphorus compounds and carbamates, such as diisopropylphosphofluoridate and eserine. The enzyme was inhibited by some compounds, such as atropine and quinidine. Auaternary ammonium salts showed an inhibitory effect on the enzyme resembling co-operative inhibition.

Recognition sequence of a restriction endonuclease from Haemophilus gallinarum.

From comparison of the sequences in and around the cleavage sites of restriction endonuclease HgaI isolated from Haemophilus gallinarum, the recognition sequence and cleavage site of this enzyme was deduced as below: (formula: see text) This enzyme recognizes a specific but asymmetric penta-nucleotide sequence, GCGTC, and introduces staggered cleavages at appointed positions away from the recognition sequence, generating protruding 5'-ends of five nucleotides. The sequences surrounding the cleavage sites bear no obvious relation to one another.

New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI).

Two new restriction endonucleases have been isolated from Flavobacterium okeanokoites IFO12536 and Micrococcus luteus IFO12992 and named FokI and MluI, respectively. Based on analysis of the sequences around the restriction sites, the recognition sequences and cleavage sites of these endonucleases were deduced as below: FokI: (formula: see text). MluI introduces double-strand cleavages at unique sequences that are completely two-fold rotationally symmetric like most type II restriction endonucleases. FokI belongs to a class of restriction endonucleases that recognize specific but asymmetric nucleotide sequences and introduce staggered cleavages at appointed positions away from the recognition sequences.

The nucleotide sequence of the cloned tufA gene of Escherichia coli.

The 4 kb (8.5 % lambda units) EcoRI fragment harboring the tufA gene of Escherichia coli was cloned using plasmid pTUA1 (Shibuya et al., 1979) and its structure was analyzed. The nucleotide sequence of about 1500 base pairs, covering the C-terminal portion of elongation factor EF-G (fus gene), the intercistronic region between fus and tufA, the entire structural gene for tufA with the GUG initiation and UAA termination codons, and the 3' flanking region of tufA, was determined. Comparison of the tufA nucleotide sequence with the tufB sequence (An and Friesen, 1980) and the known amino acid sequence of EF-Tu (Arai et al., 1980) revealed that the products of genes tufA and tufB are identical except for one amino acid at the C-terminal, i.e., glycine for tufA and serine for tufB. Nucleotide differences between tufA and tufB were found at 13 positions. Among them, one in the initiation codon and the other one in the C-terminal amino acid codon had replacements at the first letter of the codons. The other eleven changes were in the third codon positions, which did not affect the amino acid coding. The pattern of codon usage in tufA and tufB is highly nonrandom, and remarkably similar to that in ribosomal protein genes, with the codons for the most abundant species of isoaccepting tRNAs being preferentially utilized (Post et al., 1979; Post and Nomura, 1980).

Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid.

The nucleotide sequence of a 2248 bp portion of the plasmid mini-F has been determined. This region includes the replication origin and all of the plasmid-coded information required for replication. The same region is also capable of expressing incompatibility. A striking feature of the sequence is the presence of nine 19-bp repeating units. Four of these repeats, all arranged in one direction, comprise a cluster, and the remaining five, all arranged in the opposite direction, comprise another cluster. These clusters are separated by a region of about 850 bp that encodes a hypothetical 29-kd polypeptide. This region has sequences highly homologous to those found in the origin regions of the Escherichia coli (Sugimoto et al., 1979; Meijer et al., 1979) and Salmonella typhimurium (Zyskind and Smith, 1980) genomes.

A new site-specific endonuclease from Streptomyces lavendulae (SlaI).

A new restriction-like endonuclease, SlaI, was found and partially purified from Streptomyces lavendulae ATCC8664. This endonuclease cleaved bacteriophage lambda DNA at only one site, and cytosine-substituted bacteriophage T4 DNA at 16 sites. The recognition sequence was determined by using SlaI fragments of cytosine-substituted bacteriophage T4 DNA. The hexanucleotide recognized by SlaI endonuclease was (Formula: see text), with the sites of cleavage as indicated by the arrows. Therefore, SlaI endonuclease was an isoschizomer of XhoI endonuclease.

Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein.

The insecticidal crystal protein (ICP) gene, icp, from a 68-kb plasmid derived from Bacillus thuringiensis subsp. sotto was cloned in Escherichia coli. The icp expression in E. coli cells was confirmed by both immunological and insect-toxicity assays of the cell extract. The entire icp gene resides in the 6.6-kb PstI fragment, which codes for a 144-kDal peptide identical to the intact ICP, as determined by its size and reaction with anti-ICP antibody. Deletion analysis further revealed that the 2.8-kb region within the 6.6-kb PstI fragment codes for ICP. Analysis of the nucleotide sequence indicated that a peptide of 934 amino acid residues truncated at the C-terminal end is encoded by this 2.8-kb fragment. A unique feature of this truncated ICP is the abundance of cysteine and lysine residues within its C-terminal region.

DNA sequence of Crithidia fasciculata kinetoplast minicircles.

Kinetoplast DNA (kDNA) networks of the insect trypanosomatid Crithidia fasciculata strain CF-C1 contain a nearly homogeneous population of kDNA minicircles as judged by restriction enzyme cleavage analysis. We have determined the entire nucleotide sequence of the major class of minicircles by analyzing M13 phage clones carrying half-length segments of the kDNA minicircle molecules. The 12 nucleotide sequence d(G-G-G-G-T-T-G-G-T-G-T-A) is the longest sequence common to kDNA minicircles from several trypanosome species examined to date. Two copies of this universal minicircle sequence were identified 180 degrees apart as direct repeats within the C. fasciculata kDNA minicircles. In addition, these universal minicircle sequences are contained within direct repeats with nearly identical sequences of 173 and 177 base pairs (bp) in length. These sequences are also conserved in the same arrangement in minor sequence classes of minicircles from this strain. Site-specific discontinuities on both strands of the minicircle, identified previously in minicircle replication intermediates, were localized within the 173 and 177 bp conserved sequences. These sequences were also found to have extensive homology with similar conserved sequences in kDNA minicircles from Leishmania tarentolae. We suggest that the two conserved sequences, each containing a single copy of the universal minicircle sequence, represent replication origins in the Crithidia minicircles.

Serum carbohydrate-deficient transferrin in patients with nonalcoholic liver disease and with hepatocellular carcinoma.

Serum carbohydrate-deficient transferrin (CDT) is used as a reliable and specific marker of alcohol consumption. However, recent studies have shown false-positive CDT test results in nonalcoholic liver disease. We examined the clinical significance of serum CDT in nonalcoholic liver disease, especially hepatocellular carcinoma. Serum CDT was measured in 23 teetotallers, 56 patients with alcoholic liver disease, 84 patients with viral liver disease and 67 patients with hepatocellular carcinoma, with an Axis %CDT radioimmunoassay kit, and the results were expressed as percentages of the total transferrin (%CDT). The mean serum %CDT value was increased 1.8-fold in alcoholic liver fibrosis and 3.8-fold in alcoholic liver cirrhosis compared with the teetotallers. The serum %CDT values in viral chronic hepatitis were similar to those of the teetotallers, and were increased 2.0-fold in viral liver cirrhosis. False-positive results were found in 10 (37%) of the 27 patients with viral liver cirrhosis. The mean serum %CDT value was increased 2.5-fold in hepatocellular carcinoma, and false-positive results were found in 31 (46%) of the 67 patients. The serum %CDT value was related to the severity of Child grade, the size of tumor and the grade of histological differentiation. These results suggest that the ability of serum CDT test to detect chronic alcoholism may be reduced in patients with nonalcoholic liver cirrhosis and those with hepatocellular carcinoma.

The 5'-terminal region of the apocytochrome b transcript in Crithidia fasciculata is successively edited by two guide RNAs in the 3' to 5' direction.

We analyzed the chimeric guide RNA (gRNA)-mRNA molecules in Crithidia fasciculata that are predicted to transiently exist in editing of the 5'-terminal domain of apocytochrome b (CYb) mRNA, by polymerase chain reaction amplification and DNA sequencing, and obtained evidence suggesting that among the 14 editing sites numbered from 3' to 5', the sequence in the 3'-half of the sites (3' block) was specified by one guide RNA species (gRNA-I) and that in the remaining half of the sites (5' block) by the other guide RNA species (gRNA-II) and that the direction of editing in each block was 3' to 5'. The predicted transition site of editing by two gRNAs was between the first and second U residues from the 3' end within editing site 7. We found that a stretch of the edited sequence in the 3' block of mRNA could form a stable duplex with a stretch immediately upstream of the guide sequence in gRNA-II. The result leads to a successive editing model that the 3' block of pre-edited mRNA is first edited by gRNA-I, and after completion of editing, the 5' portion of gRNA-II pairs with the edited mRNA for editing of the 5' block.

Structure of viral DNA in a rat cell line transformed by the cloned EcoRI-C fragment of adenovirus 12.

A DNA segment carrying viral DNA was cloned from a rat cell line transformed by the cloned EcoRI-C fragment (0 to 16.4 map units) of human adenovirus type 12(Ad12), and the viral sequence in the clone was analysed. The cloned segment contained the region from nucleotide positions 118 to 3520 of the Ad12 genome in the middle. No unique structure was found at the viral and non-viral DNA junctions. When examined the transforming activity, the conserved viral sequence was able to transform rat 3Y1 cells efficiently. Southern blotting analysis of the viral sequence in five re-transformed cell lines showed that the viral sequence was inserted at different sites of cellular DNA. These results indicate that (I) the Ad12 DNA moiety from the enhancer-promoter region of the E1A gene to the end of the E1B gene contains enough information for efficient transformation of the rat cell, and (II) integration of the viral sequence at unique cellular sites is not prerequisite for transformation.

The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.

Phosphate in dialysis patients.

A single pool extracellular compartment (10-15 L) model was demonstrated to fit changes in plasma Pi (at steady state or rebound during HD) on the assumption that its G increased linearly or exponentially during HD. G at the end of HD was estimated to be 30-300 times that at the start of HD, which suggested rapid outflow of Pi from its reservoirs. When bovine or human RBC as a cellular model of probable Pi reservoirs were incubated with solute-free NSS or dialyzed plasma, BUN instantaneously reached a new equilibrium, but Pi and CR gradually flowed out from RBC. Pi efflux from RBC was observed even after the Pi concentration in the incubation media exceeded its initial plasma concentration. With regard to BUN and Cr, the concentration in RBC estimated from that in the incubation media decreased exponentially during HD corresponding to the change in their plasma concentrations. Estimated concentration of Pi in RBC, however, remained unchanged during HD in spite of significant decreases in its plasma concentration. These results suggest that Pi does not leave RBC by simple diffusion and that RBC contains Pi precursor(s). 31P NMR spectra obtained from packed RBC showed that uremic RBC contained more ATP and unidentified compounds (possibly sugar phosphates) than nonuremic RBC and that the peak amplitude of the latter unidentified compounds significantly decreased in uremic RBC leaving the dialyzer or at the end of HD. Furthermore, it was demonstrated that during incubation at 37 degrees C with NSS or dialyzed plasma, the peak amplitude of 2, 3-DPG decreased in contrast with an increased Pi peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic behavior of plasma phosphate in chronic dialysis patients.

1. Change in the plasma P concentration during intra- and interdialytic phases is notably different from that for BUN, CR and UA. Reduction rate for P depends on its pretreatment concentration. Plasma P is apt to level off or rebound even during a treatment and quickly returns to the pretreatment level after it is terminated. 2. In short-term evaluations, occurrence of P rebound during a treatment does not correlate with factors such as meal, A1 gel, dialyzer type, dialyzer membrane and therapeutic mode, but with the pretreatment P concentration. Once it reaches a threshold level inherent to each patient, plasma P seems to rebound. 3. Pretreatment P concentration in each patient seemed to be controlled in a relatively narrow range. 4. While apparent generation rates (G) estimated with a single compartmental kinetic model are stable during intra- and interdialytic phases as to BUN, CR and UA, G for P seems to be significantly enhanced especially during the latter period of the treatment and immediately after the termination of the treatment.

The FokI restriction-modification system. II. Presence of two domains in FokI methylase responsible for modification of different DNA strands.

Based on the previous findings that the FokI methylase (MFokI) consists of 647 amino acid residues and contains two copies of the segment specific for adenine methylase, Asp-Pro-Pro-Tyr, at amino acid positions 218-221 and 548-551, the role of these copies in the methylation reaction was investigated by introduction of a mutation into each segment. The MFokI gene was inserted into M13 vectors, and the Asp residues in the two segments were converted to Gly and Ala by oligonucleotide-directed mutagenesis. The wild-type and mutant genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the FokI recognition site was treated with each of these enzymes, and after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to FokI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by the introduction of mutations into one of the two segments, and that the segments at the N-terminal and C-terminal moieties participated in modification of the strands carrying 5'-GGATG-3' and 3'-CCTAC-5', respectively. We concluded that MFokI contained two functional domains each of which was responsible for modification of different strands in the target DNA.